Electrostatic Map of Proteasome ?-Rings Encodes the Design of Allosteric Porphyrin-Based Inhibitors Able to Affect 20S Conformation by Cooperative Binding

The importance of allosteric proteasome inhibition in the treatment of cancer is becoming increasingly evident. Motivated by this urgent therapeutic need, we have recently identified cationic porphyrins as a highly versatile class of molecules able to regulate proteasome activity by interfering with gating mechanisms. In the present study, the mapping of electrostatic contacts bridging the regulatory particles with the ?-rings of the human 20S proteasome led us to the identification of (meso-tetrakis(4-N-methylphenyl pyridyl)-porphyrin (pTMPyPP4) as a novel non-competitive inhibitor of human 20S proteasome. pTMPyPP4 inhibition mechanism implies a positive cooperative binding to proteasome, which disappears when a permanently open proteasome mutant (?-3?N) is used, supporting the hypothesis that the events associated with allosteric proteasome inhibition by pTMPyPP4 interfere with 20S gating and affect its "open-closed" equilibrium. Therefore, we propose that the spatial distribution of the negatively charged residues responsible for the interaction with regulatory particles at the ?-ring surface of human 20S may be exploited as a blueprint for the design of allosteric proteasome regulators.